Gambogic Acid Induces G0/G1 Cell Cycle Arrest and Cell Migration Inhibition Via Suppressing PDGF Receptor β Tyrosine Phosphorylation and Rac1 Activity in Rat Aortic Smooth Muscle Cells

Abstract

Gambogic acid (GA) is the major active compound of Gamboge, a brownish or orange resin exuded from Garcinia hanburryi tree in Southeast Asia. Previous studies have demonstrated that GA exhibits potent anticancer effects by inducing cell cycle arrest or apoptosis in many types of cancer cell lines and blocking angiogenesis via inhibition of vascular endothelial cell proliferation and migration. Proliferation and migration of vascular smooth muscle cells (VSMCs) are critical steps in the progress of atherosclerosis and restenosis after angioplasty. In the present study, we investigated whether GA has an inhibitory effect on the proliferation and migration of VSMCs and its possible mechanism. The inhibitory effect of GA on the proliferation induced by PDGF-BB and EGF was measured by using Cell number counting assay and [(3)H]-thymidine incorporation. The effects of GA on the cell cycle progression and viability stimulated by PDGF-BB and EGF were also analyzed by flow cytometry analysis. The inhibitory effect of GA on the migration stimulated by PDGF-BB was measured by transwell chamber assay. The effect of GA on the Cell cycle regulatory molecules (cyclinD1, cyclinE, CDK2, CDK4), PDGFR and its downstream signaling molecules including ERK1/2, PLCγ1, AKT and JNK was measured by western blotting. The effect of GA on the Rac1 activity was measured by using GST-pulldown assay. The effects of GA on the tyrosine phosphorylation stimulated by PDGF-BB and EGF and the capacity of GA binding with PDGF-BB and EGF were also measured. We found that GA significantly inhibited proliferation, migration and DNA synthesis in primary cultured rat aortic VSMCs at concentrations of 0.25, 0.5, 1.0 and 2.0 µmol/L after stimulation of 50 µg/L platelet-derived growth factor-BB (PDGF-BB). GA induced G0/G1 phase arrest in the cell cycle progression of VSMCs. No obvious necrosis or apoptosis was found with GA treatment. The expressions of CDK2, CDK4, cyclin D1 and cyclin E, cell cycle regulatory molecules, were significantly suppressed by GA treatment in a concentration-dependent manner. The phosphorylation of PDGF receptor β (PDGFR-β) and the activities of downstream intracellular signaling molecules including phospho-ERK, phospho-PLCγ1, phospho-AKT, phospho-JNK and GTP-Rac1 were also inhibited by GA pretreatment. GA inhibited PDGFR-β phosphorylation through inhibiting the activity of tyrosine directly, rather than indirectly via binding PDGF-BB. The results of this study provide preliminary evidence that the inhibitory effects of GA on VSMCs proliferation and migration may be mediated through multiple signal pathways controlled by PDGF-Rβ activation and its downstream intracelluar signaling.